The principal factor controlling blood glucose levels is the hormone insulin. Diabetes may result either from insufficient production of insulin or from resistance to the action of circulating insulin.
It has been suggested that a peptide fragment of human growth hormone (hGH), derived from enzymic digestion of pituitary, can counter the effects of natural inhibitors of insulin (Australian patent application no. 87961/75 by Choay S. A.). This .alpha.-peptide chain, having sequence leu-ser-arg-leu-phe-asp-asn-ala, is comprised of amino acids all of which have the L configuration.
This peptide, equivalent to hGH 6-13, was stated to be able to counteract the inhibition of insulin by somantin, a degradation product of growth hormone, and was thought to potentiate the effects of insulin, to induce synthesis of insulin receptor sites on cells, and to sensitize .beta. cells in the islets of Langerhans to glucose.
However, it has subsequently been found that the peptide disclosed by Choay does not have these activities. In fact, inconsistency in demonstrating the claimed biological activities, due to the absence of detailed knowledge of the precise conformational structure of the active substance, prevented further development and validation of the original work by Bornstein and co-workers, inventors in respect of 879661/75, and this application lapsed.
Synthesis of peptides has been carried out on a small scale since the initial work of Emil Fischer in 1900 and became a commonplace technique in biochemistry in the 1950s, when solution phase methods were used. The development of solid phase peptide synthetic techniques by Merrifield in the late 1960s resulted in an explosive expansion of this work, and it is now regarded as a routine technique in biochemistry. The peptides which can be constructed using these techniques may have the same sequences as naturally occurring peptides; sequences which are modified from the natural sequence at one or more points, or are entirely non-natural; sequences which incorporate amino acids which do not occur naturally in living organisms, including D-amino acids, .epsilon.-amino acids, and non-naturally occurring .alpha.-amino acids; and sequences which incorporate both amino acids and other chemical structures which are not themselves amino acids. In other words, these techniques provide the ability to modify or construct sequences at will; by these means, the structural requirements of peptides, in particular their 3-dimensional structures, which are necessary for their proper functioning have been explored.
Part of these explorations commonly includes the substitution of amino acids in a given sequence either by other amino acids, which may be naturally-occurring or non-naturally-occurring, or by analogous chemical structures; the only requirement is that such analogous chemical structures must be able to be attached to the growing peptide chain, and the analogues are usually chosen so that they can be used in conventional solid phase synthetic methods. It is well known that if such a substitution is successful with one peptide, it is likely to succeed in other situations, including other peptide sequences. The effect of a given substitution can now be predicted on a theoretical basis in small peptides, based on a limited number of experiments.
We have now surprisingly found that although .alpha.-peptide chains equivalent to hGH 6-13 are biologically inactive, peptides in which residues equivalent to asp.sub.11 and asn.sub.12 of the hGH sequence form a .beta.-imide group do have biological activity. This cyclic imide group, which forms a structure known in the art as a Type II' .beta.-turn, forms a characteristic 3-dimensional structure which we believe to be essential for activity of the peptide. The aspartimido group is well known to be associated with a Type II' .beta.-turn.
We have now prepared a family of novel .beta.-imide linked L-aspartyl-L-asparaginyl peptides wherein the general structure of the peptide may additionally involve between 2 and 6 other .alpha.-amino acid residues of either the D- or L-stereochemical form, or involve other non .alpha.-amino acid substitutions at positions 1 or 6 to 12.
Furthermore, the in vitro and in vivo properties of these .beta.-imido-L-aspartyl-L-asparaginyl peptides as potent hypoglycaemic agents with functional capabilities of potentiating insulin action are demonstrated. In this application we describe (i) the synthesis of these peptides via solid and solution phase peptide synthetic protocols; (ii) the purification and structural characterization of these peptides; (iii) the in vitro evaluation of these peptides in various biological assay systems; (iv) the evaluation of these peptides in lowering blood glucose levels in vivo.